Poor quality of platelet concentrates obtained from donors correlates with low efficiency and increased risk for adverse reactions in recipients. The circulation half-life of platelets is about 5 days after which time they become senescent i.e. undergo a number of physiological changes that leads to their ultimate removal from the circulation primarily by macrophages in the spleen and liver. In order to keep platelets in concentrates (i.e., under highly artificial conditions) alive for at least 5 days, platelet activation has to be inhibited. Calcium chelators and the pH lowering effect of anticoagulants (Bouchard et al. Interactions between platelets and the coagulation system. In: Platelets. Ed: Michelson A D.), gentle preparation techniques (Hagberg et al. Transfusion. 2000; 40(2):182-192.) and the use of biocompatible materials for tubing and containers (Iwasaki et al. J Biomed Mater Res. 2001; 57(1):72-8.) contribute to preserving platelet integrity; however, during storage platelets lose their integrity and viability, a phenomenon generally known as the platelet storage lesion (Devine et al. Transfusion. 1999; 39(7):724-734.) Some characteristics of activated fresh platelets can be found in old stored platelets such as increased P-selectin expression (Barnard et al. Transfusion. 1999; 39(8; 850-8.) and increased number of microparticles. Microparticles are either budded from the plasma membrane or released during secretion and range in size between 40 nm and 1 μm (Nieuwland R, Sturk A. Platelet-derived microparticles. In: Platelets. (editor: A D Michelson). Academic Press 2002; 255-65.). Other indicators such as responsiveness to physiologic agonists are modified in stored platelets (Curvers et al. Transfusion. 2004; 44(1):49-58.).
At present the determination of platelet quality is very laborious and time consuming because no single test exists for this purpose. Thus, a whole panel of parameters is usually investigated. These include platelet morphology scoring (Rock et al. Transfusion. 2003; 43(10):1374-7.), the expression of activation markers on the platelet surface (Holme et al. Transfusion. 1997; 37(1):12-17.) and the presence and characterization of platelet microparticles with flow cytometry (Kim et al. Blood Coagul Fibrinolysis. 2002; 13(5):393-397.), the response to hypotonic shock (Holme et al. Transfusion. 1998; 38(1):31-40.), the extent of shape change (Hunter et al. Transfusion. 2001; 41(6):809-14; Holme et al. Transfusion. 1997; 37(1):5-11.) and, more recently, the detection of platelet mitochondrial activity (Pich et al. Free Radic Res. 2002; 36(4):429-436; Perrotta et al. Transfusion. 2003; 43(4):526-35.). Manual morphology scoring on the microscope is an integral part of platelet characterization in transfusion medicine and has been proven to be one of the best in vitro tests for platelet quality (Kunicki et al. Transfusion. 1975; 15(5): 414-421.) with good correlation to in vivo survival time (Slichter S J, Harker L A. Br J Haematol. 1976; 34(3):395-402; Slichter S J, Harker L A. Br J Haematol. 1976; 34(3):403-19.). However, morphology scoring on the microscope is time consuming and subjective and accordingly is not practical for rapidly testing large batches of platelet concentrates.
Optical methods have been proposed and developed to measure platelet quality but they suffer shortcomings that prevent their use in routine quality determination. For example, the so-called swirling effect is a simple but crude and subjective test for the discoid shape of platelets in concentrates. Platelet monitoring using the swirling effect routinely in an automated device (Bellhouse et al. Br J Haematol. 1987; 66(4):503-8.) has not proven successful.
Light transmission measured in an aggregometer reflects the responsiveness of platelets to exogenous agonists (Bom G V R, Thorngren M. Brit J Radiol. 1985; 58 (693):922-923; Born G V R. Adv Exp Med Biol 1985; 192: 399-409.). While this test is easy to perform, it requires large sample volumes (0.5 mL) and a control sample for each run. Although the initial decrease in light transmission after addition of an agonist has been previously reported to reflect platelet shape change by Latimer at al. (Latimer et al. Arch Biochem Biophys. 1977; 180:151-159.), it has later been questioned (Latimer P. Appl Opt. 1983; 22:1136-1143.). It has now been shown that this signal is caused by platelet microaggregation (Maurer-Spurej E, and Devine D V. Lab Invest. 2001; 81(11): 1517-26.). If platelets are preactivated, for example by exposure to room temperature, the kinetics of aggregation are enhanced and light transmission decreases immediately (Maurer-Spurej E, and Devine D V. Lab Invest. 2001; 81(11): 1517-26.). The instrument developed by ChronoLog that utilizes the initial decrease in light transmission as an indicator of platelet quality, therefore, measures the extent of microaggregation rather than the “extent of shape change”.
Static light scattering measures the intensity of the scattered light at different scattering angles which results in an average particle size and optical density (Fratantoni et al. Lab Clin Med. 1984; 103(4):620-31.). Platelet shape change has only a minor effect on the actual platelet volume or the optical density of platelets (David et al. Coll Surface B: Biointerfaces. 1996; 6:101-14; Hubbell et al. Thromb Haemost. 1991; 65:601-7.). It has been previously shown that the static light scattering curves of two morphologically different platelet populations are therefore nearly the same (Maurer-Spurej et al. Lab Invest 2001; 81(4):581-592.). Therefore, this type of measurement is not adequate for measuring platelet quality.
Spurej et al. (Spurej et al. Experientia 1992; 48:71-79) have used dynamic light scattering (DLS) to estimate electrophoretic mobility and diffusion coefficient and obtain information about the morphology of platelets under physiological conditions. However, no correlation with platelet quality was measured and electrophoretic light scattering is difficult to implement as a routine technique. Furthermore the experiments were conducted on purified platelet samples which are not representative of platelet concentrates.
Bayer developed the H*System hematology analyzer (Zelmanovic D, Hetherington E J. Vet Clin Path. 1998; 27(1):2-9.) and the new ADVIA 120 (Bayer Diagnostics)(Zelmanovic et al. United States patent, Oct. 6, 1998, U.S. Pat. No. 5,817,519.). Both instruments measure static light scattering to determine platelet activation. Several assumptions are made: 1. The shape of platelets is spherical. 2. Platelet density decreases with activation due to the release of granule content. 3. The refractive index of a platelet is extracted from a look-up table generated with latex spheres (Chapman et al. Thromb Haemost. 2003; 89(6):1004-15.). It is quite obvious that this approach is mathematically demanding and not very flexible.
Special light scattering instruments were developed that utilize static light scattering to measure microaggregation (David et al. Coll Surface B: Biointerfaces. 1996; 6:101-14; Eto et al. Cardiovasc Res. 1998; 40(1):223-9; Tomida et al. Thromb Res 1998;92:221-228; Yabasaki K, Kokufuta E. Langmuir. 2002; 18:39-45.) taking advantage of the fact that microaggregates scatter much more light than single platelets. The Japanese company Kowa brought several versions of this instrument to the market (PA-100, PA-200). However, these instruments are not designed to measure platelet morphology.
Furthermore, while some of the parameters discussed above have been linked to platelet activation there exist no solid evidence that they correlate with platelet quality in the sense of being suitable for blood transfusion.
Therefore, no test exists to rapidly and routinely measure platelet quality and platelet concentrates are released without quality testing. About 65,000 platelet concentrates have to be discarded by Canadian Blood Services because they reach their 5-day expiry date before they can be transfused. This translates to a loss of several million dollars. Similar discard rates are encountered in major blood banks around the world. With the implementation of bacterial testing and pathogen inactivation, platelet quality remains the major determinant for the out-date of platelet concentrates.
In view of the above it would be highly desirable to have a simple and rapid method for determining platelet quality.